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MLST typing of Pasteurella multocida associated with haemorrhagic septicaemia and development of a real-time PCR specific for haemorrhagic septicaemia associated isolates Andreas Petersen a, Magne Bisgaard b, Kirsty Townsend c, Henrik Christensen a,* a
Department of Veterinary Disease Biology, Faculty of Medical and Health Sciences, University of Copenhagen, Stigbøjlen 4, 1870 Frederiksberg C, Denmark Horsevænget, Viby, Denmark c Microbiology and Immunology, School of Veterinary and Life Sciences, Murdoch University, Australia b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 December 2013 Received in revised form 12 February 2014 Accepted 14 February 2014
Two serovars of Pasteurella multocida, B:2 and E:2, have been reportedly associated with haemorrhagic septicaemia (HS), a peracute and devastating disease mainly affecting cattle and water buffaloes. We multilocus sequence typed (MLST) 64 isolates of P. multocida including 55 associated with HS and found that they mainly included sequence type (ST) 122 (n = 50) and rarely ST63 (n = 1), ST147 (n = 2) and ST162 (n = 2) compared to other members of the species isolated from other lesion types and hosts. Single-nucleotide polymorphisms suitable for specific detection of STs associated with HS were detected in the est gene. A new HS-est-RT-PCR (est indicating the target gene) specifically detected ST122, ST63, ST147 and ST162 associated with HS. The new HS-est-RT-PCR did not detect strains of ST151 with capsular type D isolated from pigs that were found positive with a previously published HS PCR detection method. The new HS-est-RT-PCR represents a fast and specific detection of the specific types of P. multocida involved in HS. The HS-est-RTPCR developed in the current study seems to more accurately identify isolates of P. multocida associated with HS compared to PCR detection methods previously published. ß 2014 Elsevier B.V. All rights reserved.
Keywords: Clonal complex ST122 Acute septicaemia Rirdc
1. Introduction Pasteurella multocida is a heterogeneous group of bacteria which has a unique capability to infect a wide range of animals resulting in a broad spectrum of diseases, like haemorrhagic septicaemia in cattle and water buffaloes, fowl cholera, atrophic rhinitis in pigs and pneumonia in ruminants, pigs and rabbits (Carter and De Alwis, 1989; Chanter and Rutter, 1989; Frank, 1989; Gilmour and Gilmour, 1989; Manning et al., 1989; Rhoades and Rimler, 1989). In spite of major differences in disease
* Corresponding author. Tel.: +45 35332783; fax: +45 35332755. E-mail address: [email protected] (H. Christensen).
manifestations certain antigenic compositions can be associated with specific diseases in animals. This is especially true for the B:2 and E:2 serovars of P. multocida (Carter and De Alwis, 1989) associated with haemorrhagic septicaemia (HS), a peracute and devastating disease characterized by oedematous swelling in the headthroat-brisket region, swollen and haemorrhagic lymph nodes and the presence of numerous subserous petechial haemorrhages. HS affects cattle and buffalo in certain areas of Asia, in the near and Middle East, and in a number of countries in Africa (Shivachandra et al., 2011). For unknown reasons serovar E:2 has only been reported from Africa, while B:2 occasionally is reported from other parts of the world than Asia and Africa. Similarly, infections in other animals than bovines are reported from time to
http://dx.doi.org/10.1016/j.vetmic.2014.02.022 0378-1135/ß 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Petersen, A., et al., MLST typing of Pasteurella multocida associated with haemorrhagic septicaemia and development of a real-time PCR specific for haemorrhagic septicaemia associated isolates. Vet. Microbiol. (2014), http://dx.doi.org/10.1016/j.vetmic.2014.02.022
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time (Carter and De Alwis, 1989). The diagnosis of HS depends on clinical signs, gross pathological lesions, morbidity and mortality and on the isolation of P. multocida from blood or bone marrow (Anon, 2012). Wilson et al. (1992) reported that all 13 serogroup E isolates from the African continent investigated had identical somatic antigens and shared the same HhaI REA profile. Using HpaII for digestion of DNA, however, separated the 13 isolates into five groups. The same authors also investigated 71 capsular type B isolates from swine, deer, cattle, birds, buffalo, elk, bison, yak and unknown sources and demonstrated different HhaI REA profiles. Townsend et al. (1997b), demonstrated a remarkable homogeneity among Asian HS isolates by ribotyping and PFGE with a clear distinction observed between virulent and avirulent HS isolates. Among isolates of P. multocida obtained from acute septicaemic pasteurellosis in Vietnamese pigs, most isolates were shown to represent capsular type B (Townsend et al., 1998b). Although diversity was observed as to somatic antigens, all isolates shared to the same REP-PCR type with three B:2 isolates from bison (USA) and buffalo (India and Vietnam). Unfortunately, the molecular methods used above only address short term epidemiology, and do not allow lab-tolab comparison. In addition, they do not allow establishing the clonal relationship between isolates from different hosts and diseases in order to understand long term epidemiology and assess the risk of transmission between different animal species. Multilocus sequence typing (MLST) is based upon determining short nucleotide sequences of several (5–7) housekeeping genes that have undergone some evolutionary diversification leading to polymorphism (Maiden et al., 1998). Using this method 39 sequence types were recognized among 63 Australian poultry isolates and the three reference strains for P. multocida (Subaaharan et al., 2010). Subsequent studies by Bisgaard et al. (2013) as well as additions in RIRDC (Rural Industries Research and Development Corporation) identified several clonal complexes within P. multocida that included capsular type B (ST50, ST62, ST63, ST122, ST129, ST163, ST164) and E (ST122, ST127, ST147, ST162). Brickell et al. (1998) reported a PCR test specific for serotype B:2 and the HSB-PCR of Townsend et al. (1998a) was found specific for serovars B:2, B:5 and B:2,5. The targets of both PCRs seem to be Mu-like prophages. The aims of the present investigation were to MLST type a diverse collection of P. multocida capsular types B and E from different animal species, and subsequently examine the phylogenetic relationship of capsular types B and E isolates originating from different animal species and lesion types to improve our basic understanding of the epidemiology of HS. In addition, the aim of the study was to develop a specific Real-Time PCR (HS-est-RT-PCR) for rapid detection of the lineage of P. multocida involved in HS. 2. Materials and methods Sixty-four strains were included from the collection of Townsend including five reference strains of P. multocida (Table 1). The PCRs of Townsend et al. (1998a, 2001) were used for initial screening of isolates.
MLST typing was performed according to Subaaharan et al. (2010) using the RIRDC database. All STs have been registered in the MLST database at RIRDC. This MLST database (http://pubmlst.org/pmultocida/) was developed by Keith Jolley and sited at the University of Oxford (Jolley et al., 2004). In addition to strains in the current study, information about 52 strains available from RIRDC were included the comparison by MLST. For sequence types in the current study and of type strains of subspecies of P. multocida obtained from RIRDC, concatenated sequences of the seven genes behind the MLST scheme were aligned and a neighbour joining phylogenetic tree was generated by ClustalX (Thompson et al., 1997) and graphically presented by MEGA4 (Tamura et al., 2007). Single-nucleotide polymorphisms were analyzed using software Minimum SNP (Price et al., 2007). A singlenucleotide polymorphism (SNP) was detected in the est gene which is one of the seven genes included in the RIRDC MLST scheme for P. multocida. Primers were designed with software Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and using allele sequences as template. A forward primer was designed to anneal in an area where all alleles were identical, while allele specific reverse primers were placed with 30 -end directly over and matching one of the polymorphisms. An intentional mismatch was incorporated in the reverse primers to increase allele specificity (Newton et al., 1989). The resulting primers were Pm HS F 50 -CCTTCTACACAAGSTGGTTTGA-30 , PmHSAR 50 -GCCAARTCATTACCRTCT-30 and PmHSTR 50 GCCAARTCATTACCRTCA-30 (Table 2) amplifying 178 bp of the est gene. The RT-PCR method is labelled HS-est-RT-PCR as a consequence of the target gene. The HS-est-RT-PCR was run with Maxima SYBR Green qPCR Master Mix (Fermentas) and primers in 0.3 mM concentration on a MxPro3000 (Stratagene). Running conditions were an initial denaturation at 95 8C for 10 min, followed by 40 cycles of denaturation at 95 8C for 30 s, annealing 50 8C for 1 min and elongation 72 8C for 15 s. Finally, a dissociation analysis was performed for 1 cycle consisting of denaturation at 95 8C for 1 min, annealing at 55 8C for 30 s and denaturation at 95 8C for 30 s. DCT values were calculated by subtracting the CT value for the matched primer/template pair from the CT of the misprimed set. CT values were obtained using the default threshold value. The primers were first tested against four isolates with known basepair at the specific site. Additional 12 isolates were subsequently randomly selected and HS-est-RT-PCR was performed. After the SNPs were resolved a full MLST sequence type was determined for the isolates (Table 1). 3. Results MLST of 64 isolates of P. multocida associated with HS in mainly bovines but also pigs and buffalo, demonstrated ST122 (n = 50), ST63 (n = 1), ST147 (n = 2) and ST162 (n = 2) (Table 1), all of which clustered together in the phylogenetic tree (Fig. 1). Nine strains isolated from nasal swabs or tonsils of apparently healthy pigs in Vietnam represented ST151 (n = 9) which clustered with ST14 of P. multocida subsp. gallicida. ST122, ST63, ST147 and ST162 had identical alleles in the genes adk, zwf, mdh and gdh.
Please cite this article in press as: Petersen, A., et al., MLST typing of Pasteurella multocida associated with haemorrhagic septicaemia and development of a real-time PCR specific for haemorrhagic septicaemia associated isolates. Vet. Microbiol. (2014), http://dx.doi.org/10.1016/j.vetmic.2014.02.022
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Table 1 Strains of Pasteurella multocida investigated in relation to haemorrhagic septicaemia (HS), sequence type (ST) and HS-est-RT-PCR detection. Strain
Serovarb
Host
Country
Year
HSB-PCRb
ST
HS-est-RT-PCR
P2252a P1234a VP131 VP133 VP142A VP144K P1248 VP286 VP289 VP585 VP588 VP655 VP656 VP657 VP132 VP135 VP141A VP143A VP145a VP163 VP164a VP230 VP231 VP237a VP247a VP249 VP250 VP251 VP252 VP253 VP326 VP327 VP328 VP329 VP330 VP331 VP332 VP333 VP334 VP347a VP557 VP558 VP559 VP560 VP586 VP589 VP593 VP652 VP653 VP654 VP243a (=O150 =2417SIe) VP582 VP583 VP584 (=P1235) P3263 VP726 VP732 VP733 VP737 VP746 VP747 VP749 VP750 VP772
B, Roberts Id Ed B B:5,16 B:5,7,8 B:2,5 NamiokaB:6 B B Bd Bd B B B B:5,16 B:2,5 B:5,6,7,16 B:5 B:5 B:2 B:2,5 B:2 B:2 B:2 B:2 B:2 B:2 B:2 B:2 B:2 B:2,5 B:2,5 B:2,5 B:2,5 B:2,5 B:2,5 B:2 B:5 B:2 B:5,6 D:1 D:1 D:1 A:1 Bd Bd Bd B B B Ed
NDc Bovine Bovine Bovine Porcine Porcine ND Buffalo Bovine Bovine ND Bovine Buffalo Porcine Bovinef Bovine Porcineg Porcine Porcine Porcineg Porcineg Bovine Porcineg Buffalog Porcineg Buffaloh Bovineg Buffalog Bovineg Buffalog Bovine Bovine Bovine Bovine Bovine Bovine Bovine Bovine Bovine Porcineg Bovine Bovine Bovine Bovine ND ND ND Buffalo Buffalo Buffalo Bovine
India Central Africa Philippines ND Vietnam Vietnam ND Malaysia Philippines Burma Iraq Vietman Vietnam Vietnam Philippines ND Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam ND Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam ND ND ND Vietnam Vietnam Vietnam Africa
ND ND ND ND ND ND ND ND ND ND 1952 ND ND ND ND ND ND ND ND ND ND ND ND 1996 1996 1997 1986 ND 1986 1986 1993 1994 1994 1994 1993 1993 1993 1993 1993 ND ND ND ND ND ND ND ND ND ND ND ND
ND + ND + + + ND + ND ND ND + + + + + ND ND + + + ND + + + + + + + + + + + + + + + + + + + + + + ND ND ND + + + +
122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 147
ND ND ND ND + ND ND + ND ND + ND ND ND + ND + ND ND ND ND + ND ND + + ND ND ND ND + ND ND ND ND ND ND ND ND ND + ND ND + ND ND ND ND ND ND +
E E E B:2,5; Little Lyon 2 D D D D D D D D D
Bovine Bovine Bovine Buffalo Porcinei Porcinei Porcinei,f Porcinei Porcinei Porcinej Porcinej Porcinej Porcinej
S. Africa Africa Central Africa ND ND ND Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam
ND ND ND ND 1999 1999 1999 1999 1999 1999 1999 1999 1999
ND ND ND ND + + + + + + + + +
147 162 162 63 151 151 151 151 151 151 151 151 151
ND + + + ND ND ND ND ND ND ND ND
a b c d e f g h i j
Included with Townsend et al. (1997a,b, 1998a,b). PCR capsular typing according to Townsend et al. (2001) and HSB-PCR according to Townsend et al. (1998a). ND, not determined or data not available. Positive for HS ELISA (Dawkins et al., 1990). Rick Rimler, Carter type E. Clinically sick animal. Heart blood. Bone marrow. Nasal swab. Tonsil.
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Table 2 Binding sites of the reverse primers (PmHSAR, PmHSTR) within the est gene sequence. Positiona Est allele 35 Est allele 37 Est allele 39 Est allele 10 Primer PmHSAR Primer PmHSTR a
366 T T T A A T
367 G G G G G G
368 G G G G A A
369 C C C T Y Y
370 G G G G G G
371 G G G G G G
372 T T T T T T
373 A A A A A A
374 A A A A A A
375 T T T T T T
376 G G G G G G
377 A A A A A A
378 T T T T Y Y
379 T T T T T T
380 T T T T T T
381 G G G G G G
382 G G G G G G
383 C C C C C C
Position in the DNA sequence of the est gene of strain Pm70 accession no. AE004439.
the nine ST151 strains all tested positive with the specific HSB-PCR (Fig. 2). The four STs found to be associated with HS in the present paper formed a clonal complex (ST63, ST122, ST147 and ST162) and were related to ST127 (single locus variant of ST162) that was reported in RIRDC only represented by a single bovine African isolate of serovar E (Table 3). Although variations occurred in this clone with respect to one or two variant alleles generating other STs, ST122 was by far the dominant type, while the other STs are only represented by a few isolates. When the concatenated sequences were compared at the level of the whole RIRDC database, the five STs again turned out to be the closest related with 99.6 (ST122, ST27) up to 100% (ST122, ST63). The 100% similarity relates to only 1 nt difference in the est gene.
ST63 is a single locus variant of ST122 (est gene) and ST162 a double locus variant of ST122 (est and pmi genes). ST147 is a double locus variant to ST162 (pmi and pgi genes). Comparison to isolates with ST122 in RIRDC showed recent deposition from India and other South East Asian countries of isolates associated with HS (Table 3). ST147 and ST162 were only demonstrated in Africa (Table 1). Minimum SNP detected one singlenucleotide polymorphism in the est gene, where the HS associated allele demonstrated a T-A basepair, while all other STs demonstrated an A-T basepair (Table 2). The new HS-est-RT-PCR specifically detected 15 strains of ST122, ST63, ST147 and ST162 randomly selected and tested, while ST151 tested negative (Table 1). The nine isolates of ST151 all had capsular type D compared to mainly B and E in the other four STs (Table 1). However,
ST122
95
ST63
68
93 93
HS clonal complex
ST162
99
ST127 ST147
85
ST29 67
ST13 (including strain NCTC 10322T of P. multocida subsp. multocida) ST9 (including strain Pm70 of P. multocida) 98
ST62
46
ST51 ST129
23 54
ST151 99
18
ST50 ST14 (including strain NCTC 10204T of P. multocida subsp. gallicida) ST164
28 94
ST163 100
ST54
ST15 (including strain CIP A125T of P. multocida subsp. septica)
ST8
0.001
Fig. 1. Neighbour joining phylogenetic tree of the 7 concatenated gene sequences used for the MLST of STs investigated and reference strains. Bootstrap percentages are shown at internal branches. The scale bar represents sequence variation considering the model for nucleotide substitution (Jukes & Cantor) and tree-shape used in the neighbour joining analysis.
Please cite this article in press as: Petersen, A., et al., MLST typing of Pasteurella multocida associated with haemorrhagic septicaemia and development of a real-time PCR specific for haemorrhagic septicaemia associated isolates. Vet. Microbiol. (2014), http://dx.doi.org/10.1016/j.vetmic.2014.02.022
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Table 3 Sequence types (STs) of isolates of P. multocida obtained from bovines and pigs with lesions related to haemorrhagic septiceamia (HS) and with capsular serovars B and E. Data are from the current study and compared to data in the RIRDC (Rural Industries Research and Development Corporation) database. ST
Isolates current
Isolates RIRDC
Pig
Bovine, sheep
Other
NI
Lesion
Country/continent
Capsular serovar
Reference
122
43
56
11
81
2
5
HS
B, E
Hotchkiss et al. (2011), Bisgaard et al. (2013), and Moustafa et al. (2013)
63 162 147 127 151 8b 51c
0 1 0 0 6 0 0
2 1 2 1 3 1 1
0 0 0 0 9 0 0
2 2 2 1 0 1 3
0 0 0 0 0 0 0
0 0 0 0 0 0 0
NIa NI NI NI NI NI HS
Africa, Burma, East Timor, Iraq, Malaysia, Philippines, Vietnam, India, Indonesia, Pakistan, Sri Lanka, Thailand NI Africa Vietnam, S. Africa Africa,Vietnam NI NI NI
B E E E D NIb A
Bisgaard et al. (2013)
129d 50
0 0
1 18
0 13
1 1
0 3
0 1
Sri Lanka Czeck Republic, Denmark, Germany, UK
B A, B, D
62
0
3
0
3
0
0
New Zealand
B
163 164
0 0
1 1
0 0
0 0
1 1
0 0
HS Pneumonia, atrophic rhinitis, bronchopneumonia Pneumonia, pleuritis and periotonitis NI NI
USA USA
B B
a b c d
Hotchkiss et al. (2011) Hotchkiss et al. (2011) Hotchkiss et al. (2011) and Bisgaard et al. (2013) Hotchkiss et al., 2011 Hotchkiss et al. (2011) and Bisgaard et al. (2013) McFadden et al. (2011)
NI, no information. 33 other isolates not associated with HS. 2 other isolates of bovine origin without information of disease. 43 other isolates not associated with HS.
Fig. 2. Agarose gel electrophoresis of HSB-PCR products of strains of ST151 (see Table 1) isolated form pigs. The positive control was strain VP133 (see Table 1) and the negative control, C was without DNA. The size marker, M was GeneRuler 1 kb Plus (ThermoScientific). The nucleotide sizes of the HSB-PCR product as well as of M are indicated.
4. Discussion Previous investigations of P. multocida have found serotyping unreliable as to investigating a possible relationship between serovars and lesion types due to
problems with untypeable isolates, cross reactions and the lack of relationship between genotype and serovar (Wilson et al., 1993; Singh et al., 2013). The isolates from HS included four STs of capsular types B or E. Most B types belonged to Heddleston type 2,5 although other variants
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were found. Surprisingly, three D:1 and a single A:1 serotype isolated from bovines also belonged to ST122. This observation is in accordance with Shivachandra et al. (2011), who listed ten other serotypes besides B:2 and E:2 to have been isolated from HS outbreaks. The five strains of capsular type E from Africa belonged to ST122, ST147 and ST162, indicating that these strains belong to different but related STs. Based on MLST, strains of capsular type E cannot be separated from capsular type B. Ninety-nine isolates have been allocated to ST122 including 43 from the current study (Table 3). The 56 isolates from RIRDC included isolates from a recent publication by Moustafa et al. (2013). The related ST147 and ST162 are from the current study whereas ST63 was included with the publication of Bisgaard et al. (2013) (Table 3). This indicates a clonal population of P. multocida to be associated with HS in agreement with Cardoso-Toset et al. (2013), who showed three STs from bovine HS to be closely related to a fourth ST of porcine source by using the Multiple host MLST database (http://pubmlst.org/ pmultocida_multihost/). Unfortunately further comparison between the two databases with respect to the HS clonal complex cannot be done since common reference strains are not included. The HS clonal complex of P. multocida that included five closely related ST and dominated by ST122 showed some relationships on single allele level to other STs associated with ruminants, but rarely with pigs. The closest related outside the clonal complex was ST29 with 99.5%. It shared all but two alleles with the complex (zwf and mdh) and was only represented by a single isolate from chicken. Outside the clonal complex, allele 23 of adk was only found in ST96, ST98 and ST203, all including bovine isolates of Carter serovar A. Out of the three est alleles (35, 37, 39) present in the clonal complex, only 35 was found in another ST (ST120) that included a pig isolate from pneumonia. The pmi allele 17 was also found in ten other STs (ST92, ST93, ST94, ST95, ST99, ST100–ST104) all of which included bovine isolates located with a ‘ruminant’ cluster in Bisgaard et al. (2013). The relationship at single allele level but not at ST level indicates recombination rather than point mutation as a dominant population genetic mechanism that has generated these STs. For each ST at least one strain was tested by HS-est-RTPCR. The MLST already indicated that all isolates in each ST shared identical sequences, and for the same reason additional testing of isolates within each ST by HS-est-RTPCR was not necessary. The new HS-est-RT-PCR reported, represents a fast and specific test for detection of the specific STs of P. multocida involved in HS. The new HS-estRT-PCR did not detect pig isolates representing a different phylogenetic line (ST151) compared to the previous HSBPCR of Townsend et al. (1998a), however, it identified ten porcine strains of ST122 in agreement with Townsend et al. (1998b), who occasionally found the same genotype of P. multocida to be isolated from pigs and bovines with acute septicaemia and HS, respectively. Problems with the definition of HS may also lead to problems with accurate detection by the HS-est-RT-PCR. As mentioned in the introduction five conditions have to be satisfied for HS: clinical signs, gross pathological lesions
(oedematous swelling in the head-throat-brisket region, swollen and haemorrhagic lymph nodes and the presence of numerous subserous petechial haemorrhages), morbidity and mortality and on the isolation of P. multocida from blood or bone marrow (Anon, 2012). Whereas the isolation of P. multocida is always satisfied with respect to a positive PCR test, especially the gross pathological lesions could have been mistaken in the first place. This could be the explanation for the lack of detection of ST8, ST51 and ST129 with the HS-est-RT-PCR. For all three STs only a single isolate (BCC 2256, BCC 344 and BCC 2009) out of a total of 34, 3 and 44 isolates, respectively (see Table 3) were specifically associated with HS. The same three isolates were the only ones not belonging to ST122 in Hotchkiss et al. (2011). Brickell et al. (1998) reported all capsular type B of Heddleston type 2 (when available) to be detected by their PCR in addition to one strain of serovar E, whereas one B and two E strains were not detected. Strains typed as Roberts type I and Namioka type 6B were all detected similar to the observation in the current study with the HSest-RT-PCR developed. The HSB-PCR of Townsend et al. (1998a) only tested capsular type B of Heddleston types 2 or 5 or 2,5 positive, but did not test strains of capsular type E or other capsular types as positive. The current investigation further showed that this PCR occasionally can test capsular type D as positive that cannot directly be associated with acute septicaemia in pig (see Table 1). The HS-est-RT-PCR developed in the current study therefore seems to more accurately identify isolates of P. multocida associated with HS compared to previous PCRs of Brickell et al. (1998) and Townsend et al. (1998a). In conclusion, MLST rather than serotyping is recommended for verification of cases of HS since both serovars D and A strains were shown to belong to the clonal complex associated with HS. Due to porcine isolates of serovar D from apparently healthy pigs were confirmed positive in the HS-PCR of Townsend et al. (1998a), we also recommend that isolates from lesions of typical HS are characterized by both the new HS-est-RT-PCR and the HS-PCR of Townsend et al. (1998a) to investigate the correlation between the clonal complex associated with HS and the results of these two PCRs. Acknowledgements Pia Mortensen and Dan F. Ryttov are thanked for excellent technical assistance. The project was funded by the Danish Research Council for Technology and Production Sciences. References Anon, 2012. Haemorrhagic septicaemia. OIE terrestrial manual 2008, 1– 13 (Chapter 2.4.12)The World Organisation for Animal Health (OIE), Paris. http://www.oie.int/ Bisgaard, M., Petersen, A., Christensen, H., 2013. Multilocus sequence analysis of Pasteurella multocida demonstrates a type species under development. Microbiology 159, 580–590. Brickell, S.K., Thomas, L.M., Long, K.A., Panaccio, M., Widders, P.R., 1998. Development of a PCR test based on a gene region associated with the pathogenicity of Pasteurella multocida serotype B:2, the causal agent of haemorrhagic septicaemia in Asia. Vet. Microbiol. 59, 295–307.
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VETMIC-6524; No. of Pages 7 Veterinary Microbiology xxx (2014) xxx–xxx
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MLST typing of Pasteurella multocida associated with haemorrhagic septicaemia and development of a real-time PCR specific for haemorrhagic septicaemia associated isolates Andreas Petersen a, Magne Bisgaard b, Kirsty Townsend c, Henrik Christensen a,* a
Department of Veterinary Disease Biology, Faculty of Medical and Health Sciences, University of Copenhagen, Stigbøjlen 4, 1870 Frederiksberg C, Denmark Horsevænget, Viby, Denmark c Microbiology and Immunology, School of Veterinary and Life Sciences, Murdoch University, Australia b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 December 2013 Received in revised form 12 February 2014 Accepted 14 February 2014
Two serovars of Pasteurella multocida, B:2 and E:2, have been reportedly associated with haemorrhagic septicaemia (HS), a peracute and devastating disease mainly affecting cattle and water buffaloes. We multilocus sequence typed (MLST) 64 isolates of P. multocida including 55 associated with HS and found that they mainly included sequence type (ST) 122 (n = 50) and rarely ST63 (n = 1), ST147 (n = 2) and ST162 (n = 2) compared to other members of the species isolated from other lesion types and hosts. Single-nucleotide polymorphisms suitable for specific detection of STs associated with HS were detected in the est gene. A new HS-est-RT-PCR (est indicating the target gene) specifically detected ST122, ST63, ST147 and ST162 associated with HS. The new HS-est-RT-PCR did not detect strains of ST151 with capsular type D isolated from pigs that were found positive with a previously published HS PCR detection method. The new HS-est-RT-PCR represents a fast and specific detection of the specific types of P. multocida involved in HS. The HS-est-RTPCR developed in the current study seems to more accurately identify isolates of P. multocida associated with HS compared to PCR detection methods previously published. ß 2014 Elsevier B.V. All rights reserved.
Keywords: Clonal complex ST122 Acute septicaemia Rirdc
1. Introduction Pasteurella multocida is a heterogeneous group of bacteria which has a unique capability to infect a wide range of animals resulting in a broad spectrum of diseases, like haemorrhagic septicaemia in cattle and water buffaloes, fowl cholera, atrophic rhinitis in pigs and pneumonia in ruminants, pigs and rabbits (Carter and De Alwis, 1989; Chanter and Rutter, 1989; Frank, 1989; Gilmour and Gilmour, 1989; Manning et al., 1989; Rhoades and Rimler, 1989). In spite of major differences in disease
* Corresponding author. Tel.: +45 35332783; fax: +45 35332755. E-mail address: [email protected] (H. Christensen).
manifestations certain antigenic compositions can be associated with specific diseases in animals. This is especially true for the B:2 and E:2 serovars of P. multocida (Carter and De Alwis, 1989) associated with haemorrhagic septicaemia (HS), a peracute and devastating disease characterized by oedematous swelling in the headthroat-brisket region, swollen and haemorrhagic lymph nodes and the presence of numerous subserous petechial haemorrhages. HS affects cattle and buffalo in certain areas of Asia, in the near and Middle East, and in a number of countries in Africa (Shivachandra et al., 2011). For unknown reasons serovar E:2 has only been reported from Africa, while B:2 occasionally is reported from other parts of the world than Asia and Africa. Similarly, infections in other animals than bovines are reported from time to
http://dx.doi.org/10.1016/j.vetmic.2014.02.022 0378-1135/ß 2014 Elsevier B.V. All rights reserved.
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time (Carter and De Alwis, 1989). The diagnosis of HS depends on clinical signs, gross pathological lesions, morbidity and mortality and on the isolation of P. multocida from blood or bone marrow (Anon, 2012). Wilson et al. (1992) reported that all 13 serogroup E isolates from the African continent investigated had identical somatic antigens and shared the same HhaI REA profile. Using HpaII for digestion of DNA, however, separated the 13 isolates into five groups. The same authors also investigated 71 capsular type B isolates from swine, deer, cattle, birds, buffalo, elk, bison, yak and unknown sources and demonstrated different HhaI REA profiles. Townsend et al. (1997b), demonstrated a remarkable homogeneity among Asian HS isolates by ribotyping and PFGE with a clear distinction observed between virulent and avirulent HS isolates. Among isolates of P. multocida obtained from acute septicaemic pasteurellosis in Vietnamese pigs, most isolates were shown to represent capsular type B (Townsend et al., 1998b). Although diversity was observed as to somatic antigens, all isolates shared to the same REP-PCR type with three B:2 isolates from bison (USA) and buffalo (India and Vietnam). Unfortunately, the molecular methods used above only address short term epidemiology, and do not allow lab-tolab comparison. In addition, they do not allow establishing the clonal relationship between isolates from different hosts and diseases in order to understand long term epidemiology and assess the risk of transmission between different animal species. Multilocus sequence typing (MLST) is based upon determining short nucleotide sequences of several (5–7) housekeeping genes that have undergone some evolutionary diversification leading to polymorphism (Maiden et al., 1998). Using this method 39 sequence types were recognized among 63 Australian poultry isolates and the three reference strains for P. multocida (Subaaharan et al., 2010). Subsequent studies by Bisgaard et al. (2013) as well as additions in RIRDC (Rural Industries Research and Development Corporation) identified several clonal complexes within P. multocida that included capsular type B (ST50, ST62, ST63, ST122, ST129, ST163, ST164) and E (ST122, ST127, ST147, ST162). Brickell et al. (1998) reported a PCR test specific for serotype B:2 and the HSB-PCR of Townsend et al. (1998a) was found specific for serovars B:2, B:5 and B:2,5. The targets of both PCRs seem to be Mu-like prophages. The aims of the present investigation were to MLST type a diverse collection of P. multocida capsular types B and E from different animal species, and subsequently examine the phylogenetic relationship of capsular types B and E isolates originating from different animal species and lesion types to improve our basic understanding of the epidemiology of HS. In addition, the aim of the study was to develop a specific Real-Time PCR (HS-est-RT-PCR) for rapid detection of the lineage of P. multocida involved in HS. 2. Materials and methods Sixty-four strains were included from the collection of Townsend including five reference strains of P. multocida (Table 1). The PCRs of Townsend et al. (1998a, 2001) were used for initial screening of isolates.
MLST typing was performed according to Subaaharan et al. (2010) using the RIRDC database. All STs have been registered in the MLST database at RIRDC. This MLST database (http://pubmlst.org/pmultocida/) was developed by Keith Jolley and sited at the University of Oxford (Jolley et al., 2004). In addition to strains in the current study, information about 52 strains available from RIRDC were included the comparison by MLST. For sequence types in the current study and of type strains of subspecies of P. multocida obtained from RIRDC, concatenated sequences of the seven genes behind the MLST scheme were aligned and a neighbour joining phylogenetic tree was generated by ClustalX (Thompson et al., 1997) and graphically presented by MEGA4 (Tamura et al., 2007). Single-nucleotide polymorphisms were analyzed using software Minimum SNP (Price et al., 2007). A singlenucleotide polymorphism (SNP) was detected in the est gene which is one of the seven genes included in the RIRDC MLST scheme for P. multocida. Primers were designed with software Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and using allele sequences as template. A forward primer was designed to anneal in an area where all alleles were identical, while allele specific reverse primers were placed with 30 -end directly over and matching one of the polymorphisms. An intentional mismatch was incorporated in the reverse primers to increase allele specificity (Newton et al., 1989). The resulting primers were Pm HS F 50 -CCTTCTACACAAGSTGGTTTGA-30 , PmHSAR 50 -GCCAARTCATTACCRTCT-30 and PmHSTR 50 GCCAARTCATTACCRTCA-30 (Table 2) amplifying 178 bp of the est gene. The RT-PCR method is labelled HS-est-RT-PCR as a consequence of the target gene. The HS-est-RT-PCR was run with Maxima SYBR Green qPCR Master Mix (Fermentas) and primers in 0.3 mM concentration on a MxPro3000 (Stratagene). Running conditions were an initial denaturation at 95 8C for 10 min, followed by 40 cycles of denaturation at 95 8C for 30 s, annealing 50 8C for 1 min and elongation 72 8C for 15 s. Finally, a dissociation analysis was performed for 1 cycle consisting of denaturation at 95 8C for 1 min, annealing at 55 8C for 30 s and denaturation at 95 8C for 30 s. DCT values were calculated by subtracting the CT value for the matched primer/template pair from the CT of the misprimed set. CT values were obtained using the default threshold value. The primers were first tested against four isolates with known basepair at the specific site. Additional 12 isolates were subsequently randomly selected and HS-est-RT-PCR was performed. After the SNPs were resolved a full MLST sequence type was determined for the isolates (Table 1). 3. Results MLST of 64 isolates of P. multocida associated with HS in mainly bovines but also pigs and buffalo, demonstrated ST122 (n = 50), ST63 (n = 1), ST147 (n = 2) and ST162 (n = 2) (Table 1), all of which clustered together in the phylogenetic tree (Fig. 1). Nine strains isolated from nasal swabs or tonsils of apparently healthy pigs in Vietnam represented ST151 (n = 9) which clustered with ST14 of P. multocida subsp. gallicida. ST122, ST63, ST147 and ST162 had identical alleles in the genes adk, zwf, mdh and gdh.
Please cite this article in press as: Petersen, A., et al., MLST typing of Pasteurella multocida associated with haemorrhagic septicaemia and development of a real-time PCR specific for haemorrhagic septicaemia associated isolates. Vet. Microbiol. (2014), http://dx.doi.org/10.1016/j.vetmic.2014.02.022
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Table 1 Strains of Pasteurella multocida investigated in relation to haemorrhagic septicaemia (HS), sequence type (ST) and HS-est-RT-PCR detection. Strain
Serovarb
Host
Country
Year
HSB-PCRb
ST
HS-est-RT-PCR
P2252a P1234a VP131 VP133 VP142A VP144K P1248 VP286 VP289 VP585 VP588 VP655 VP656 VP657 VP132 VP135 VP141A VP143A VP145a VP163 VP164a VP230 VP231 VP237a VP247a VP249 VP250 VP251 VP252 VP253 VP326 VP327 VP328 VP329 VP330 VP331 VP332 VP333 VP334 VP347a VP557 VP558 VP559 VP560 VP586 VP589 VP593 VP652 VP653 VP654 VP243a (=O150 =2417SIe) VP582 VP583 VP584 (=P1235) P3263 VP726 VP732 VP733 VP737 VP746 VP747 VP749 VP750 VP772
B, Roberts Id Ed B B:5,16 B:5,7,8 B:2,5 NamiokaB:6 B B Bd Bd B B B B:5,16 B:2,5 B:5,6,7,16 B:5 B:5 B:2 B:2,5 B:2 B:2 B:2 B:2 B:2 B:2 B:2 B:2 B:2 B:2,5 B:2,5 B:2,5 B:2,5 B:2,5 B:2,5 B:2 B:5 B:2 B:5,6 D:1 D:1 D:1 A:1 Bd Bd Bd B B B Ed
NDc Bovine Bovine Bovine Porcine Porcine ND Buffalo Bovine Bovine ND Bovine Buffalo Porcine Bovinef Bovine Porcineg Porcine Porcine Porcineg Porcineg Bovine Porcineg Buffalog Porcineg Buffaloh Bovineg Buffalog Bovineg Buffalog Bovine Bovine Bovine Bovine Bovine Bovine Bovine Bovine Bovine Porcineg Bovine Bovine Bovine Bovine ND ND ND Buffalo Buffalo Buffalo Bovine
India Central Africa Philippines ND Vietnam Vietnam ND Malaysia Philippines Burma Iraq Vietman Vietnam Vietnam Philippines ND Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam ND Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam ND ND ND Vietnam Vietnam Vietnam Africa
ND ND ND ND ND ND ND ND ND ND 1952 ND ND ND ND ND ND ND ND ND ND ND ND 1996 1996 1997 1986 ND 1986 1986 1993 1994 1994 1994 1993 1993 1993 1993 1993 ND ND ND ND ND ND ND ND ND ND ND ND
ND + ND + + + ND + ND ND ND + + + + + ND ND + + + ND + + + + + + + + + + + + + + + + + + + + + + ND ND ND + + + +
122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 122 147
ND ND ND ND + ND ND + ND ND + ND ND ND + ND + ND ND ND ND + ND ND + + ND ND ND ND + ND ND ND ND ND ND ND ND ND + ND ND + ND ND ND ND ND ND +
E E E B:2,5; Little Lyon 2 D D D D D D D D D
Bovine Bovine Bovine Buffalo Porcinei Porcinei Porcinei,f Porcinei Porcinei Porcinej Porcinej Porcinej Porcinej
S. Africa Africa Central Africa ND ND ND Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam
ND ND ND ND 1999 1999 1999 1999 1999 1999 1999 1999 1999
ND ND ND ND + + + + + + + + +
147 162 162 63 151 151 151 151 151 151 151 151 151
ND + + + ND ND ND ND ND ND ND ND
a b c d e f g h i j
Included with Townsend et al. (1997a,b, 1998a,b). PCR capsular typing according to Townsend et al. (2001) and HSB-PCR according to Townsend et al. (1998a). ND, not determined or data not available. Positive for HS ELISA (Dawkins et al., 1990). Rick Rimler, Carter type E. Clinically sick animal. Heart blood. Bone marrow. Nasal swab. Tonsil.
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Table 2 Binding sites of the reverse primers (PmHSAR, PmHSTR) within the est gene sequence. Positiona Est allele 35 Est allele 37 Est allele 39 Est allele 10 Primer PmHSAR Primer PmHSTR a
366 T T T A A T
367 G G G G G G
368 G G G G A A
369 C C C T Y Y
370 G G G G G G
371 G G G G G G
372 T T T T T T
373 A A A A A A
374 A A A A A A
375 T T T T T T
376 G G G G G G
377 A A A A A A
378 T T T T Y Y
379 T T T T T T
380 T T T T T T
381 G G G G G G
382 G G G G G G
383 C C C C C C
Position in the DNA sequence of the est gene of strain Pm70 accession no. AE004439.
the nine ST151 strains all tested positive with the specific HSB-PCR (Fig. 2). The four STs found to be associated with HS in the present paper formed a clonal complex (ST63, ST122, ST147 and ST162) and were related to ST127 (single locus variant of ST162) that was reported in RIRDC only represented by a single bovine African isolate of serovar E (Table 3). Although variations occurred in this clone with respect to one or two variant alleles generating other STs, ST122 was by far the dominant type, while the other STs are only represented by a few isolates. When the concatenated sequences were compared at the level of the whole RIRDC database, the five STs again turned out to be the closest related with 99.6 (ST122, ST27) up to 100% (ST122, ST63). The 100% similarity relates to only 1 nt difference in the est gene.
ST63 is a single locus variant of ST122 (est gene) and ST162 a double locus variant of ST122 (est and pmi genes). ST147 is a double locus variant to ST162 (pmi and pgi genes). Comparison to isolates with ST122 in RIRDC showed recent deposition from India and other South East Asian countries of isolates associated with HS (Table 3). ST147 and ST162 were only demonstrated in Africa (Table 1). Minimum SNP detected one singlenucleotide polymorphism in the est gene, where the HS associated allele demonstrated a T-A basepair, while all other STs demonstrated an A-T basepair (Table 2). The new HS-est-RT-PCR specifically detected 15 strains of ST122, ST63, ST147 and ST162 randomly selected and tested, while ST151 tested negative (Table 1). The nine isolates of ST151 all had capsular type D compared to mainly B and E in the other four STs (Table 1). However,
ST122
95
ST63
68
93 93
HS clonal complex
ST162
99
ST127 ST147
85
ST29 67
ST13 (including strain NCTC 10322T of P. multocida subsp. multocida) ST9 (including strain Pm70 of P. multocida) 98
ST62
46
ST51 ST129
23 54
ST151 99
18
ST50 ST14 (including strain NCTC 10204T of P. multocida subsp. gallicida) ST164
28 94
ST163 100
ST54
ST15 (including strain CIP A125T of P. multocida subsp. septica)
ST8
0.001
Fig. 1. Neighbour joining phylogenetic tree of the 7 concatenated gene sequences used for the MLST of STs investigated and reference strains. Bootstrap percentages are shown at internal branches. The scale bar represents sequence variation considering the model for nucleotide substitution (Jukes & Cantor) and tree-shape used in the neighbour joining analysis.
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Table 3 Sequence types (STs) of isolates of P. multocida obtained from bovines and pigs with lesions related to haemorrhagic septiceamia (HS) and with capsular serovars B and E. Data are from the current study and compared to data in the RIRDC (Rural Industries Research and Development Corporation) database. ST
Isolates current
Isolates RIRDC
Pig
Bovine, sheep
Other
NI
Lesion
Country/continent
Capsular serovar
Reference
122
43
56
11
81
2
5
HS
B, E
Hotchkiss et al. (2011), Bisgaard et al. (2013), and Moustafa et al. (2013)
63 162 147 127 151 8b 51c
0 1 0 0 6 0 0
2 1 2 1 3 1 1
0 0 0 0 9 0 0
2 2 2 1 0 1 3
0 0 0 0 0 0 0
0 0 0 0 0 0 0
NIa NI NI NI NI NI HS
Africa, Burma, East Timor, Iraq, Malaysia, Philippines, Vietnam, India, Indonesia, Pakistan, Sri Lanka, Thailand NI Africa Vietnam, S. Africa Africa,Vietnam NI NI NI
B E E E D NIb A
Bisgaard et al. (2013)
129d 50
0 0
1 18
0 13
1 1
0 3
0 1
Sri Lanka Czeck Republic, Denmark, Germany, UK
B A, B, D
62
0
3
0
3
0
0
New Zealand
B
163 164
0 0
1 1
0 0
0 0
1 1
0 0
HS Pneumonia, atrophic rhinitis, bronchopneumonia Pneumonia, pleuritis and periotonitis NI NI
USA USA
B B
a b c d
Hotchkiss et al. (2011) Hotchkiss et al. (2011) Hotchkiss et al. (2011) and Bisgaard et al. (2013) Hotchkiss et al., 2011 Hotchkiss et al. (2011) and Bisgaard et al. (2013) McFadden et al. (2011)
NI, no information. 33 other isolates not associated with HS. 2 other isolates of bovine origin without information of disease. 43 other isolates not associated with HS.
Fig. 2. Agarose gel electrophoresis of HSB-PCR products of strains of ST151 (see Table 1) isolated form pigs. The positive control was strain VP133 (see Table 1) and the negative control, C was without DNA. The size marker, M was GeneRuler 1 kb Plus (ThermoScientific). The nucleotide sizes of the HSB-PCR product as well as of M are indicated.
4. Discussion Previous investigations of P. multocida have found serotyping unreliable as to investigating a possible relationship between serovars and lesion types due to
problems with untypeable isolates, cross reactions and the lack of relationship between genotype and serovar (Wilson et al., 1993; Singh et al., 2013). The isolates from HS included four STs of capsular types B or E. Most B types belonged to Heddleston type 2,5 although other variants
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were found. Surprisingly, three D:1 and a single A:1 serotype isolated from bovines also belonged to ST122. This observation is in accordance with Shivachandra et al. (2011), who listed ten other serotypes besides B:2 and E:2 to have been isolated from HS outbreaks. The five strains of capsular type E from Africa belonged to ST122, ST147 and ST162, indicating that these strains belong to different but related STs. Based on MLST, strains of capsular type E cannot be separated from capsular type B. Ninety-nine isolates have been allocated to ST122 including 43 from the current study (Table 3). The 56 isolates from RIRDC included isolates from a recent publication by Moustafa et al. (2013). The related ST147 and ST162 are from the current study whereas ST63 was included with the publication of Bisgaard et al. (2013) (Table 3). This indicates a clonal population of P. multocida to be associated with HS in agreement with Cardoso-Toset et al. (2013), who showed three STs from bovine HS to be closely related to a fourth ST of porcine source by using the Multiple host MLST database (http://pubmlst.org/ pmultocida_multihost/). Unfortunately further comparison between the two databases with respect to the HS clonal complex cannot be done since common reference strains are not included. The HS clonal complex of P. multocida that included five closely related ST and dominated by ST122 showed some relationships on single allele level to other STs associated with ruminants, but rarely with pigs. The closest related outside the clonal complex was ST29 with 99.5%. It shared all but two alleles with the complex (zwf and mdh) and was only represented by a single isolate from chicken. Outside the clonal complex, allele 23 of adk was only found in ST96, ST98 and ST203, all including bovine isolates of Carter serovar A. Out of the three est alleles (35, 37, 39) present in the clonal complex, only 35 was found in another ST (ST120) that included a pig isolate from pneumonia. The pmi allele 17 was also found in ten other STs (ST92, ST93, ST94, ST95, ST99, ST100–ST104) all of which included bovine isolates located with a ‘ruminant’ cluster in Bisgaard et al. (2013). The relationship at single allele level but not at ST level indicates recombination rather than point mutation as a dominant population genetic mechanism that has generated these STs. For each ST at least one strain was tested by HS-est-RTPCR. The MLST already indicated that all isolates in each ST shared identical sequences, and for the same reason additional testing of isolates within each ST by HS-est-RTPCR was not necessary. The new HS-est-RT-PCR reported, represents a fast and specific test for detection of the specific STs of P. multocida involved in HS. The new HS-estRT-PCR did not detect pig isolates representing a different phylogenetic line (ST151) compared to the previous HSBPCR of Townsend et al. (1998a), however, it identified ten porcine strains of ST122 in agreement with Townsend et al. (1998b), who occasionally found the same genotype of P. multocida to be isolated from pigs and bovines with acute septicaemia and HS, respectively. Problems with the definition of HS may also lead to problems with accurate detection by the HS-est-RT-PCR. As mentioned in the introduction five conditions have to be satisfied for HS: clinical signs, gross pathological lesions
(oedematous swelling in the head-throat-brisket region, swollen and haemorrhagic lymph nodes and the presence of numerous subserous petechial haemorrhages), morbidity and mortality and on the isolation of P. multocida from blood or bone marrow (Anon, 2012). Whereas the isolation of P. multocida is always satisfied with respect to a positive PCR test, especially the gross pathological lesions could have been mistaken in the first place. This could be the explanation for the lack of detection of ST8, ST51 and ST129 with the HS-est-RT-PCR. For all three STs only a single isolate (BCC 2256, BCC 344 and BCC 2009) out of a total of 34, 3 and 44 isolates, respectively (see Table 3) were specifically associated with HS. The same three isolates were the only ones not belonging to ST122 in Hotchkiss et al. (2011). Brickell et al. (1998) reported all capsular type B of Heddleston type 2 (when available) to be detected by their PCR in addition to one strain of serovar E, whereas one B and two E strains were not detected. Strains typed as Roberts type I and Namioka type 6B were all detected similar to the observation in the current study with the HSest-RT-PCR developed. The HSB-PCR of Townsend et al. (1998a) only tested capsular type B of Heddleston types 2 or 5 or 2,5 positive, but did not test strains of capsular type E or other capsular types as positive. The current investigation further showed that this PCR occasionally can test capsular type D as positive that cannot directly be associated with acute septicaemia in pig (see Table 1). The HS-est-RT-PCR developed in the current study therefore seems to more accurately identify isolates of P. multocida associated with HS compared to previous PCRs of Brickell et al. (1998) and Townsend et al. (1998a). In conclusion, MLST rather than serotyping is recommended for verification of cases of HS since both serovars D and A strains were shown to belong to the clonal complex associated with HS. Due to porcine isolates of serovar D from apparently healthy pigs were confirmed positive in the HS-PCR of Townsend et al. (1998a), we also recommend that isolates from lesions of typical HS are characterized by both the new HS-est-RT-PCR and the HS-PCR of Townsend et al. (1998a) to investigate the correlation between the clonal complex associated with HS and the results of these two PCRs. Acknowledgements Pia Mortensen and Dan F. Ryttov are thanked for excellent technical assistance. The project was funded by the Danish Research Council for Technology and Production Sciences. References Anon, 2012. Haemorrhagic septicaemia. OIE terrestrial manual 2008, 1– 13 (Chapter 2.4.12)The World Organisation for Animal Health (OIE), Paris. http://www.oie.int/ Bisgaard, M., Petersen, A., Christensen, H., 2013. Multilocus sequence analysis of Pasteurella multocida demonstrates a type species under development. Microbiology 159, 580–590. Brickell, S.K., Thomas, L.M., Long, K.A., Panaccio, M., Widders, P.R., 1998. Development of a PCR test based on a gene region associated with the pathogenicity of Pasteurella multocida serotype B:2, the causal agent of haemorrhagic septicaemia in Asia. Vet. Microbiol. 59, 295–307.
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